Vaccine against viral hepatitis and process

ABSTRACT

A VACCINE AGAINST VIRAL HEPATITIS IS DERIVED FROM BLOOD CONTAINING AUSTRALIA ANTIGEN, HAVING PARTICLES RESEMBLING VIRUSES WHICH ARE SUBSTANTIALLY FREE FROM NULELIC ACID, OF A SIZE RANGE OF 180 TO 210 A., SUBSTANTIALLY FREE FROM INFECTIOUS PARTICLES. THE VACCINE EHERE REQUIRED IS ATTENUATED OR ALTERED. THE PREFERRED PROCEDURE FOR REMOVING IMPURTIES INCLUDING INFECTIOUS COMPONENTS INVOLVES CENTRIFUGATION, ENZYME DIGESTION, COLUMN GEL FILTRATION, DIFFERENTIAL DENSITY CENTRIFUGATION IN A SOLUTION OF SUCROSE, DIALYSIS, DIFFERENTIAL DENSITY CENTRIFUGATION IN A SOLUTION OF CESIUM CHLORIDE, AND DIALYSIS.

1972 B. s. BLUMBERG ETAL 3,636,191

VACCINE AGAINST VIRAL HEPATITIS AND PROCESS Filed Oct. 1969 9Sheets-Sheet l FRACTION NUMBER Baruch S. Blumberg Irving Millman WQQCQNWW allow:

Jun. 18, 1972 a 5. BLUMBERG ETAL 3,636,191

VACCINE AGAINST VIRAL HEPAIITIS AND PROCESS FIG. 2

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Jan. 18, 1-972 B. s. BLUMBERG ETAL 5 VACCINE AGAINST VIRAL HEPATITIS ANDPROCESS Filed Oct. 8, 1969 9 Sheets-Sheet i FIG. 5

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VACCINE AGAINST VIRAL HEPATITIS AND PRDGESS Filed Oct. 84 1969 9SheetsSheet 8 FIG. 6

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VACCINE AGAINST VIRAL HEPAIITIS AND PROCESS 9 Sheets-Sheet 9 Filed Oct.

FIG.

Baruch S. Blumberg Irving Millman United States Patent i it 3,636,191Patented Jan. 18, 1972 3,636,191 VACCINE AGAINST VIRAL HEPATITIS ANDPROCESS Baruch S. Blumberg, Glenside, and Irving Millman, Willow Grove,Pa., assignors to The Institute for Cancer Research. Philadelphia, Pa.

Filed Oct. 8, 1969, Ser. No. 864,788 Int. Cl. A61k 27/00 US. Cl. 424-899 Claims ABSTRACT OF THE DISCLOSURE A vaccine against viral hepatitis isderived from blood containing Australia antigen, having particlesresembling viruses which are substantially free from nucleic acid, of asize range of 180 to 210 A., substantially free from infectiousparticles. The vaccine where required is attenuated or altered. Thepreferred procedure for removing impurities including infectiouscomponents involves centrifugation, enzyme digestion, column gelfiltration, differential density centrifugation in a solution ofsucrose, dialysis, differential density centrifugation in a solution ofcesium chloride, and dialysis.

DISCLOUSURE OF INVENTION The invention relates to a vaccine againstviral hepatitis and a process of preparation thereof.

A purpose of the invention is to remove a blood material, preferablyplasma, from an animal, suitably a human being, whose blood containsAustralia antigen and to substantially remove from the Australia antigenall normal blood constituents and infectious components and then toattenuate (alter) any residual infectious components which might bepresent in the Australia antigen fraction.

A further purpose is to remove impurities including infectiouscomponents from the Australia antigen fraction by enzyme digestion.

A further purpose is to remove impurities including infectiouscomponents from the Australia antigen fraction by differential densitycentrifugation, suitably in a solution of sucrose and separately in asolution of cesium chloride.

A further purposes is to remove impurities including infectiouscomponents from the Australia antigen fraction by centrifugation, enzymedigestion, column gel filtration, differential density centrifugation ina solution of sucrose, dialysis, differential density centrifugation ina solution of cesium chloride, and dialysis.

Further purposes appear in the specification and in the claims.

The drawings are diagrams, useful in explaining the invention.

FIG. 1 plots the result of gel filtration, plotting percent transmissionat 280 m with respect to fraction number.

FIG. 2 shows immunoelectrophoretic patterns of fractions at differentstages of purification. Pattern 1 is for starting plasma; pattern 2 isfor pellet suspensions; pattern 3 is for the enzyme treated pelletsuspension; pattern 4 is for the enzyme treated pellet suspension passedthrough gel filtration by Sephadex G200. The trough contained horseanti-human serum antiserum. v

FIG. 3 is a sucrose gradient profile, plotting Cerenkov CMP-Backgroundagainst fraction number.

FIG. 4 is a density gradient showing a composite graph of the sucroseseparated Au( 1) fraction on cesium chloride plotting absorbancy at 280m as one ordinate and Cerenkov CPM-Background as another ordinate, and

Density in grams per ml. as one abscissa and fraction number as anotherabscissa.

FIG. 5 shows immunoelectrophoretic patterns of purified Au(1) fractions15 to 18 obtained in FIG. 4. Pattern 1 shows the Au(1) band (see arrow)developed by placing anti-Au( 1) in the trough below. Pattern 2 showsthe result of reacting Au(1) with anti total human serum protein placedin the trough below. Pattern 3 shows the result when whole plasmacontaining Au(1) reacts with anti-human serum antiserum in the troughabove.

FIG. 6 is an immunodiffusion pattern of purified Au(1) fractions 15 to18 from FIG. 4. In the experiment the central well contained horse antitotal human serum antiserum. Wells 1 and 2 contained purified Au(1) (5l. and 20 l. respectively). Wells 3 and 5 contained other fractions ofplasma isolated in earlier stages of the purification scheme.

FIG. 7 shows the immunodilfusion pattern of the Au(1) fraction aftervarious treatments. The center well contained human anti-Au(1)antiserum. Wells 2 and 3 contained the Au(1) fraction heated at C. forone hour (5 and 20 l. respectively). Well 4 contained ether treatedAu(1); well 5 contained chloroform treated Au(1); wells 1 and 6contained the Au(1) fraction heated at 85 C. for one hour (5 and 20 l.respectively).

FIG. 8 shows immunodiifusion patterns of the Au(1) fraction aftervarious treatments. The protocol was the same as in FIG. 7 except thatthe center well contained rabbit anti-Au(1) antiserum.

FIG. 9 shows particles of the most purified fraction negatively stainedand at a magnification of 300,000 diameters under the electronmicroscope.

The nature of Australia antigen Australia antigen, which is designatedgenerally Au and specifically in respect to the material under study asAu(1), was first detected by Blumberg, Bull. NY. Acad. Med. 40, 377(1964), in the serum of an Australian aborigine. Its geographicaldistribution is widespread, since it is present in the blood of a largeportion of the populations of the Pacific Islands, Southeast Asia, Indiaand in many other tropical areas. It is also present in the blood ofmany institutionalized mongoloids (Downs syndrome). Au is also presentin the blood of many patients who suffer from viral hepatitis. Thegeographical distribution, disease association, genetics and physicaland chemical characteristics of Au have been reviewed by Blumberg,Sutnick and London, Bull. NY. Acad. Med. 44, 1566 (1968).

The occurrence of Australia antigen in acute and chronic hepatitis hasalso been described by Blumberg, Gerstley, Hungerford, London andSutnick, Ann. Int. Med. 66, 924 (1967); Sutnick, London, Gerstley,Crunlund and Blumberg, J. Am. Med. Assoc. 205, 670 (1968); Sutnick,London and Blumberg, Am. J. Digest, Des. 14, 189 (1969); Blumberg,Sutnick and London, J. Am. Med. Assoc. 207, 1895 1969); and London,Sutnick and Blumberg, Ann. Int. Med. 70, 55 (1969).

Initial studies have revealed the presence of particles of a size of to210 A., which are specifically agglutinated in the serum of patients whohave viral hepatitis. Bayer, Blumberg and Werner, Nature 218, 1057(1968).

The ability to stain intranuclear granules in liver cells of hepatitispatients with the same antisera coupled wtth fluorescein shows thatthese cells contain a common antigen with serum particles. Millman andBlumberg US. patent application Ser. No. 852,930, filed Aug. 25, 1969for Reagent and Test for Hepatitis; Millman, Zavatone, Gerstley andBlumberg, Nature 222, 181 (1969').

Vaccine The virus causing hepatitis exhibits some of the unusualproperties Which are also exhibited by Australia antigen. For exampleboth have a diameter less than 260 A., both survive exposure to 56 for30 minutes and both survive freezing at minus C. to minus C. for atleast one year. Kissling, Transmission of Virus by the Water Route(Inter-science Publishers, John Wyley & Sons, 1965) 337. The causativeagent must be present in the blood of affected individuals in highconcentration or must be of high infectivity since small amounts of thisblood if transferred by needle puncture can cause infection. The same istrue of Australia antigen.

We have discovered that Australia antigen from blood can be purified byremoving blood components and substantially all infectious components,out of excess of caution Australia antigen can be attenuated (altered)to remove any residual infectious components and it can be administeredto human beings as a vaccine which will protect some people againstviral hepatitis, we believe by causing the formation of antibodies inthe blood of some of the persons who receive the vaccine.

Our experience indicates that Australia antigen isolated from blood isabnormally low in, or free from nucleic acids, although we do not relyon this theory. Australia antigen appears to be the shell of a viruswhich causes hepatitis minus its nucleic acids. While Australia antigenin some cases has associated with it some infectious particles whichwill correspond to the shell plus nucleic acid, the infectious particlesare normally present in minor proportions.

Source of Australia antigen While Australia antigen can be found in theblood of some non-human primates, it is best to obtain it from the bloodof a human being in which it is present. It is best, however, not to usea patient suffering from active hepatitis but to employ a patient havingAustralia antigen in his blood, but able to tolerate it without seriousinfectious symptoms. A large proportion of the population in tropicalcountries and many persons suffering from Downs syndrome are in thiscategory.

It is preferred to use plasma as a source of Australia antigen ratherthan whole blood. Plasma may be removed by plasmaphoresis or othermethods.

Purification of Australia antigen One of the important discoveriesincident to the present invention is that Australia antigen is stronglyresistant to enzyme digestion, whereas human blood cells and otherplasma components can be reduced to peptides and removed withoutimpairing the Austrialia antigen. In this operation it is believed thatsome of the infectious components are eliminated.

Another important aspect of the present invention is that impuritiesincluding infectious components can be eliminated from the Australiaantigen fraction by difi'erential density centrifugation, which can becarried out in a solution of cesium chloride, and preferably also in aseparate solution of sucrose, the solvent in both cases being water.

As a result of these purification techniques a relatively concentratedvery pure suspension of Australia antigen in a medium such as salinesolution, substantially free from impurities including infectiouscomponents can be obtained by the invention. This purified andrelatively concentrated suspension of Australia antigen is suitable foruse as a vaccine against hepatitis. As a precaution, however, againstthe possibility that some residuum of infectious components may remain,the vaccine is attenuated or altered as later explained.

4 PREFERRED TECHNIQUE FOR PURIFICATION Sedimentation The serum obtainedby plasmaphoresis is subjected to high centrifugal force in anultracentrifuge. One hundred ml. of plasma is clarified bycentrifugation for one hour at 2000 g in a Sorvall refrigeratedcentrifuge and then re-centrifuged at 370,000 g for 18 hours in a Spinco65 Ti angle rotor ultracentrifuge. Only the pellet at the bottom of thetube contained Au(1). The remaining liquid was removed by decantationand the pellet was resuspended to 10 ml. of 0.85% (w./v.) weight ofsodium chloride solution in water.

Enzyme digestion To 10 m1. of the Au(1) suspension just referred to in a30 ml. stoppered bottle, 400 l. of amylase at a weight concentration of10 mg./ml., 400 1. of lipase at a weight concentration of 1 mg./ ml. and200 l. neuraminidase at a weight concentration of 1 mg./ml. were added.This mixture was incubated at 37 C. with gentle agitation for 15minutes, then al. of phospholipase C at a weight concentration of 1mg./ml. were added. The incubation and agitation were continued for 20minutes. During this period it was noted that the mixture gelled. To thegel was added 2 ml. of pronase at 10 rug/ml. weight concentration andthe mixture was incubated and agitated for one hour. At the end of thisperiod the gel had been completely dissolved and an additional 2 ml. ofpronase of the same concentration was added. The incubation andagitation were continued for an additional hour and then the mixture wascooled in an ice bath.

While a specific preferred procedure is given, it will be evident thatany of a wide variety of enzymes may be used in the procedure referredto above. Among the enzymes which are suitable are trypsin, pronase,lipase, phospholipase, ribonuclease, deoxyribonuclease, amylase,neuraminidase, wheat germ lipase and alpha amylase. Any other suitableenzyme may be employed if desired.

Gel filtration Five ml. aliquots of the enzyme treated suspension abovewere subjected to gel filtration through a column of preferably SephadexG-200 produced by Pharmacia, Uppsala, Sweden. This column retains thesmaller units but allows the heavier Au to come through quickly. Itworks by analogy to a mechanical sieve. Salts and inorganic materialsare also retarded. The Au comes through in the first peak.

The column was 650 mm. long and 32 mm. in diameter and it had beenpreviously equilibrated with 0.85% by weight sodium chloride solution inwater. The elution was carried on with the same solution and all of theAu(1) positive material was eluted in the peak 1 as shown in FIG. 1.

After the separation by electrophoresis as shown in FIG. 2 the patternswere devolped by adding horse anti human serum antiserum to the troughs.The decrease in the number and intensity of precipitin bands of theproduct should be noted in pattern 3 and 4. All of the fractions whichcomprise the first peak which contained all of the Au(1) antigenicitywere combined.

Sucrose gradient The combined fractions from peak 1 obtained from thegel filtration were dialyzed against 0.01 M potassium chloride solutionin water, lyophilized (freeze dried) and subjected to sedimentation onsucrose gradients in an ultracentrifuge. In carrying out this operationthe tube of the ultracentrifuge is filled with progressive increments ofsucrose solution, the most concentrated being at the bottom and theleast concentrated being at the top. The specimen resuspended indistilled water is layered on top of the differential densityincrements. The usual range of sucrose concentration in the bottom layeris about 30% (W./v.) and the sucrose concentration in the top layer isabout (w./v.) but other differential concentratial concentrations can beused. If desired an automatic machine well known in the art can supplythe sucrose gradient. For test purposes the Au(l) had been renderedradioactive prior to the initial operation and FIG. 3 shows an analysisof the gradient for F incorporation and antigenicity. Those fraction 18to 23 inclusive with major Cerenkov radioactivity and antigenicity werecombined.

As shown in FIG. 3, the line within the radioactivity peak representsAu(1) positive range when assayed by immunodiffusion. The sucrosegradients were prepared and sampled by the method of Martin and Ames, J.Biol. Chem. 236, 1372 (1961). One ml. of material was layered over eachof the two 32.5 ml. linear sucrose gradients from 10 to 30% by volume in0.01 M tris maleate buffer at a pH of 7.4. The gradients werecentrifuged in a SW 27 Spinco rotor at 57,000 Xg for hours at 2 to 5 C.Forty drop fractions were removed by puncturing the bottom of the tubesand radioactivity and antigenicity of each fraction was determined asdescribed below.

It is preferred to use a zonal ultracentrifuge for this purpse and forother differential density centrifugations as later explained but astandard ultracentrifuge may be used if desired.

While sucrose differential density centrifugation is desirable inpurifying the vaccine according to the inven tion, it is not essentialand this step can be eliminated if desired.

The active fraction after differential density centrifugation in sucrosesolution was dialyzed and lyophilized to remove the sucrose andconcentrate the Au.

Centrifugation in cesium chloride The Au from differential densitycentrifugation above were subjected to sedimentation on cesium chloridegradients. The pooled immunoreactive fractions from the sucrose densitytubes of FIG. 3 were dialyzed against 0.01 M. potassium chloridesolution in Water at 5 C. for 18 hours and lyophilized. The dried Au(l)fraction Was then resuspended in a small amount of distilled water andmixed with saturated cesium chloride solution to produce a final densityof 1.3 determined by a refractometer. The final volume of the mixturewas 5 ml. This was centrifuged in a Spinco SW 50 rotor at 222,000 g toequilibrium in about 40 hours. Ten drop fractions were collected bypuncturing the bottom of the tube.

Instead of using the technique described, a preformed linear cesiumchloride density gradient either in a centrifuge tube or a zonal rotormay be used. At the end of the centrifugation the Au bands at a certainlevel as determined by assay. The infectious particles are denser andseparate at a lower level.

FIG. 4 shows an analysis of the gradient for F incorporation, 280 mabsorption, density, and antigenicity. Antigenicity (presence of Au(l))was spread between fractions 15 and 21 with greatest activity infraction 16. The relative activity of each fraction was determined by 2fold dilution titration. That fraction which could be diluted thefarthest and produce a precipitin line was considered the peak fraction.Fraction 16 has a density of 1.21 and it falls within both the 280 mgand the Cerenkov peaks. The 280 m peak seen in FIG. 4 fraction 8, is anunknown protein that does not separate on sucrose gradient alone andcontains no Au(l) antigenicity. Fractions 15 to 18 were pooled, dialyzedagainst 0.01 M solution of potassium chloride in water and lyophilized.

FIG. 9 is a photomicrograph of Au(l) contained in the most purifiedAustralia antigen fraction.

ATTENUATION OR ALTERATION FIGS. 5 and 6 show the results of analysis ofthe pooled antigenic fractions 15 and 18 obtained by differentialdensity centrifugation in cesium chloride. There is complete absence ofreactivity with horse anti-human serum in FIG. 5, pattern 2, comparedwith the starting whole plasma of pattern 3. The presence of Au(l) isseen in pattern 1 (at the arrow) in which the purified material wastested with human anti-Au(1). The precipitin band appears to be in thesame position and has the same configuration as the band formed by theunpurified Au(l) (not shown). This indicates resistance of the antigento the previous enzyme treatment and gradient manipulations. In FIG. 6the purified fractions 15 to 18 are seen to contain no human componentswhen tested by immunodiffusion since wells 1 and 2 contained this fraction pool and there is no line of precipitation when reacted with thecentral well containing horse anti-human antiserum. Peripheral wells 3and 5 contain other Au(l) fractions from earlier stages of purificationfor comparison.

An absorption spectrum analysis of purified Au(l) reveals only asuggestion of absorbing material at 260 m There appears to be a shoulderat 275 mg and prominent peaks at 280 m and 290 mg.

The product produced as described above when suspended in a suitablemedium such as physiological saline solution is an effective vaccineagainst viral hepatitis.

In order to be sure that the vaccine is safe it should be subjected tocontrol testing, suitably using tissue culture assay or other assay toestablish that no infectious material is present. Infectivity should bedetermined both before and after attenuation or modification. The testanimals of choice are the marmoset, chimpanzee, African green (Vervet)or squirrel monkey.

The control test requires that a biopsy sample from the liver beobtained and the presence or absence of infection be determined eitherby the fluorescent technique referred to above or by any standardhistological technique.

Even though control assays establish the absence of infectiouscomponents, as an extra precaution the vaccine may be subjected toattenuation, which term as used herein includes what is sometimes calledalteration. Any well known technique for attenuation or alteration of avaccine may be employed, among which the following are suggested:

(a) Formaldehyde solution is added in progressively increasingquantities. This procedure is preferred. Samples are removed after eachaddition of formaldehye and tissue culture or other assay is employed,until infectious components are eliminated. An initial concentration offormaldehye may be 0.1 M and suitable increments up to 0.7 M may beadded. Time of contact and pH of reaction are important and would haveto be determined for each batch since these factors are empirical.Solution of phenol (0.5%) or thimerosal (Merthiolate, Lilly) (ll0,000)in combination with aging at temperatures of 2-10 C. or varying periodsof time may be substituted until infectious components are eliminated.

(b) The vaccine is subjected to ultraviolet irradiation and at the endof each exposure a sample is removed for assay until no infectiousproperties are exhibited. The exposure time for each batch is empiricaland would have to be determined.

(c) ,8 propiolactone in increments may be added and samples removedafter each addition for assay until no infectious properties exist.

(d) The vaccine may be subjected to heat treatment at graduallyincreasing temperature and a sample removed for assay after eachincrease in temperature. When a sample ceases to exhibit any infectiousproperties the heat treatment will cease. Tests show that the vaccineloses its antigenicity activity after treatment at C. or C. for one hourbut is stable at 56 C. for one hour. The heat treatment temperature evenfor a short time should not exceed 85 C.

The test for potency of the vaccine may be made in any suitable host,such as the rabbit, guinea pig, mouse or monkey. The test is positivewhen Au(l) antibody is induced.

7 DOSAGE, ADMINISTRATION AND UTILIZATION Any dosage and method ofadministration may be used which will cause the vaccine to enter theblood stream and act effectively on the immune mechanism of the body tocause the production of antibodies.

The vaccine may be enhanced by the incorporation of adjuvants such asaluminum hydroxide, aluminum phosphate or others commonly known as theArts.

The vaccine of the invention can be administered intramuscularly orsubcutaneously or by other suitable means. Intravenous administration isnot preferred because of the possible presence of pyrogens in thevaccine. Intramuscular administration is preferable.

While the vaccine may be administered to persons generally, it isrecommended particularly for persons subjected to high risk of viralhepatitis, including transfusion and infectious hepatitis. These personsinclude soldiers, sailors and other persons living in tropical areas orother areas Where hepatitis is common. The vaccine is particularlyrecommended for those in closely associated organizations, includingprisons, Downs syndrome patients, drug addicts, patients suffering fromleprosy or liable to leprosy, physicians and attendants in hospitals andinstitutions, especially those for mentally retarded patients, personslikely to receive numerous blood transfusions, such as those sufferingfrom hemophilia or thalassemia. The vaccine is recommended for moregeneral use in case of an epidemic of viral hepititis.

Resistance of Au(1) to enzyme A serum fraction containing Au(1) andparticles purified by the method of Alter and Blumberg, Blood 27, 297(1966), was standardized to a concentration which produced a sharpprecipitin line when assayed by the immuno diffusion technique.Twenty-five ,ul. portions of this fraction were reacted with a series ofenzymes under the optimal conditions described by the suppliers of theenzymes (Worthington Biochemical Corporation). The enzymes includetrypsin, wheat germ lipase, neuraminidase, alpha amylase, phospholipaseC, ribonuclease and deoxyribonuclease. In addition pronase obtained fromCalbiochemical Corporation was dissolved in distilled water at aconcentration by weight of 2 mg./ml. and ,ul. of this enzyme solutionwas reacted with 25 pl. of Au(1) of the Au(1) fraction. All enzymereactions were carried out at 37 C. for one hour.

In contrast with others antigens which have been tested and which weredegraded by these enzymes, this treatment did not damage Au(1) and issuitable as a method of purification.

Resistance to other agents The effect of elevated temperatures on Au(1)was determined by incubating equal volumes of Au(1) and 0.1 M trismaleate buffer at pH 7.5 at temperature of 56 C., 85 C. and 100 C. forone hour. The effect of elevated temperatures is shown in FIGS. 7 and 8.The heat treatment at 85 C. and 100 C. destroyed any detectableantigenicity while /4 of the amount (5 ,ul.) of untreated controlproduced a precipitin band. No effect was observed after heating at 56C. for 1 hour and a line of identity (not shown) with the untreatedcontrol was observed.

The effect of ether and chloroform on Au(1) was determined in the samemanner but at room temperature for 1 hour. The solvents were removed byevaporation in a vacuum dessicator. Both ether and chloroform treatmentsmay have uncovered antigen groups since two bands appear where oneappeared previously in FIGS. 7 and 8. One of the precipitin bands on theether treatment of the Au( 1) fraction appears to be different from oneof the two bands seen following the chloroform treatment of the Au(1)fraction in FIG. 8, since they cross and do not form a line of identity.It will be evident that other organic solvents or surface action agentsmay be useful in the purification and/or attenuation procedures.

Phenol extraction and gradient centrifugation The small amount of F inthe most purified fractions does not appear to be associated withnucleic acids of high molecular weight. To the combined fractions 15 to18 obtained by differential density centrifugation in cesium chloridewas added 2 mg. of calf thymus deoxyribonucleic acid as a carrier. Afterextraction with phenol by the procedure of Thomas and Abelson,Procedures in Nucleic Acid Research (Harper and Row 1966) p. 557 andcentrifugation in cesium chloride gradient, no radioactivity wasdetected in the density region corresponding to nucleic acids.Fluorometric assay of the most purified fractions indicated less than 1%of deoxyribonucleic acid. Assay for ribonucleic acid indicated an amountless than the lower limit of the sensitivity of the assay which wouldcorrespond to less than 10% of the weight of the total protein.

DISCUSSION AND RESULTS It is very evident that enyzme digestion does notaffect Australia antigen as determined by both immunologicalcharacteristics and by examination under the electron microscope. Theremarkable stability of Au( 1) after such treatment makes it possible toremove human plasma protein as a preliminary step. The absence of serumproteins in the vaccine has been demonstrated by the lack ofimmunological activity with human serum antiserum as determined byimmunoeletrophoresis and by immunodiffusion analysis.

The appearance of the particles in cesium chloride gradient fractionsunder the electron microscope does not differ from that reported byBayer, Blumberg and Werner, Nature, 218, 1057 (1968).

The particles have a diameter of to 210 A. and resemble viruses in theiruniformity of size. The particles appear to contain sub-units andcentral cores. The size and shape of Au(1) particles are similar tothose of the adeno associated (AAV) parvo and picornavirus groups. 1Fenner, Biology of Animal Viruses (Academic Press, 1968) 12.

In the most purified fraction there is no evidence of the presence ofany significant amount of nucleic acid. No antigenic material orparticles under the electron microscope were detected in other cesiumchloride fractions.

The density of the cesium chloride gradient peak which contained themaximum radioactivity and Australia antigen immunodiifusion activity was1.21 confirming the work of Alter and Blumberg, Blood 27, 297 (1966).The low density of purified Au(1) may be due to the lack of nucleicacids and/or the presence of lipids. The presence of lipids in Australiaantigen has been suspected due to the staining reaction of Australiaantigen precipitin bands. Alter and Blumberg, Blood 27, 297 (1966). Itis contemplated that treatment of the purified Australia antigen byorganic solvents or surface active agents may in crease the density dueto the removal of lipids.

The chemical and physical properties of Au(1), the lack of nucleic acid,and the high concentrations found in the sera of patients with viralhepititis indicate that the majority of Au(1) particles are incompleteviruses or capsids.

It will be evident that the principles of the invention are applicableto producing vaccines from other specifications, strains, varieties,variants, and types of An.

In view of our invention and disclosure, variations and modifications tomeet individual whim or particular need will doubtless become evident toothers skilled in the art, to obtain all or part of the benefits of ourinvention without copying the process and composition shown, and we.therefore, claim all such insofar as they fall within the reasonablespirit and scope of our claim.

Having thus described our invention what we claim as new and desire tosecure by Letters Patent is:

1. A vaccine against viral hepatitis essentially consist ing ofAustralia antigen, having particles substantially free from nucleicacids, of a size range of 180 to 210 A., being substantially free frominfectious particles, having a density of approximately 1.21 and beingin an amount sufficient to produce antibodies, the balance being amedium which is physiologically acceptable.

2. A vaccine of claim 1, essentially free of blood components other thanAustralia antigen.

3. A process of producing a pure vaccine against viral hepatitis, whichcomprises substantially removing impurities including infectiouscomponents from blood material containing Australia antigen by a processincluding as a major feature differential density centrifugation in asolution of cesium chloride, and adding a physiologically acceptablevehicle.

4. A process of claim 3, which includes as a precautionary feature astep adapted to attenuate any virus that might remain after the stepdirected to subsanial removal of impuriies has been completed.

5. A process of producing a pure vaccine against viral hepatitis, whichcomprises substantially removing impurities including infectiouscomponents from blood material including Australia antigen by a processincluding as a major feature enzyme digestion, and adding aphysiologically acceptable vehicle.

6. A process of producing a pure vaccine against viral hepatitis, whichcomprises substantially removing impurities including infectiouscomponents from blood material including Australia antigen bycentrifugation, enzyme digestion, column gel filtration, differentialdensity centrifugation in a solution of sucrose, dialysis, differential10 density centrifugation in a solution of cesium chloride, anddialysis, and adding a physiologically acceptable vehicle.

7. A process of producing pure vaccine against viral hepatitis, whichcomprises substantially removing impurities including infectiouscomponents from blood material containing Australia antigen by enzymedigestion, attenuating any remaining infectious components, and adding apharmaceutically acceptable vehicle.

8. A process of producing a pure vaccine against viral hepatitis, whichcomprises substantially removing impurities including infectiouscomponents from blood material containing Australia antigen by enzymedigestion, differential density centrifugation in a solution of sucrose,differential density centrifugation in a solution of cesium chloride,attenuating any remaining infectious components and incorporating thevaccine with a pharmaceutically acceptable vehicle.

9. A process of producing a pure vaccine against viral hepatitis, whichcomprises substantially removing impurities including infectiouscomponents from blood material containing Australia antigen bycentrifugation, enzyme digestion, column gel filtration, differentialdensity centrifugation in a solution of sucrose, dialysis, differentialdensity centrifugation in a solution of cesium chloride, and dialysis,attenuating any remaining infectious components and incorporating thevaccine with a pharmaceutically acceptable vehicle.

References Cited Bayer et al.: Nature, vol. 218, pages l0571059, June15, 1968.

RICHARD L. HUFF, Primary Examiner

